Glutamate racemases (EC 5.1.1.3) catalyze the cofactor-independent stereoinversion of D-and Lglutamate and are important for viability in several Gram-negative and -positive bacteria. As the only enzyme involved in the stereoinversion of L-to D-glutamate for peptidoglycan biosynthesis, glutamate racemase is an attractive target for the design of antibacterial agents. However, the development of competitive tight-binding inhibitors has been problematic and highly species specific. Despite a number of recent crystal structures of cofactor-independent epimerases and racemases, cocrystallized with substrates or substrate analogues, the source of these enzymes' catalytic power and their ability to acidify the Cα of amino acids remains unknown. The present integrated computational and experimental study focuses on the glutamate racemase from Bacillus subtilis (RacE). A particular focus is placed on the interaction of the glutamate carbanion intermediate with RacE. Results suggest that the reactive form of the RacE-glutamate carbanion complex, vis-à-vis proton abstraction from Cα, is significantly different than the RacE-D-glutamate complex on the basis of the crystal structure and possesses dramatically stronger enzyme-ligand interaction energy. In silico and experimental site-directed mutagenesis indicates that the strength of the RacE-glutamate carbanion interaction energy is highly distributed among numerous electrostatic interactions in the active site, rather than being dominated by strong hydrogen bonds. Results from this study are important for laying the groundwork for discovery and design of high-affinity ligands to this class of cofactor-independent racemases.

Glutamate racemase activity in Bacillus anthracis is of significant interest with respect to chemotherapeutic drug design, because L-glutamate stereoisomerization to D-glutamate is predicted to be closely associated with peptidoglycan and capsule biosynthesis, which are important for growth and virulence, respectively. In contrast to most bacteria, which harbor a single glutamate racemase gene, the genomic sequence of B. anthracis predicts two genes encoding glutamate racemases, racE1 and racE2. To evaluate whether racE1 and racE2 encode functional glutamate racemases, we cloned and expressed racE1 and racE2 in Escherichia coli. Size exclusion chromatography of the two purified recombinant proteins suggested differences in their quaternary structures, as RacE1 eluted primarily as a monomer, while RacE2 demonstrated characteristics of a higher-order species. Analysis of purified recombinant RacE1 and RacE2 revealed that the two proteins catalyze the reversible stereoisomerization of L-glutamate and D-glutamate with similar, but not identical, steady-state kinetic properties. Analysis of the pH dependence of L-glutamate stereoisomerization suggested that RacE1 and RacE2 both possess two titratable active site residues important for catalysis. Moreover, directed mutagenesis of predicted active site residues resulted in complete attenuation of the enzymatic activities of both RacE1 and RacE2. Homology modeling of RacE1 and RacE2 revealed potential differences within the active site pocket that might affect the design of inhibitory pharmacophores. These results suggest that racE1 and racE2 encode functional glutamate racemases with similar, but not identical, active site features.

Smooth muscle cell (SMC) degradation of the extracellular matrix and migration to the intima are fundamental processes in the vascular response to injury. NADPH oxidase-derived reactive oxygen species (ROS) are involved in development of vascular disease; however, the specific contribution of Nox1 and Nox4, the primary catalytic subunits of NADPH oxidase in SMC, is poorly understood. We hypothesized that Nox1-derived ROS mediate thrombin-dependent activation of matrix metalloproteinase 9 (MMP-9) and migration of SMCs. Studies were performed in SMCs cultured from the aorta of Nox1 null and littermate control mice. Thrombin (2 U/mL) increased superoxide levels in control SMCs, as measured by dihydroethidium, and this response was inhibited by the flavoenzyme inhibitor diphenylene iodonium (DPI, 10 mM). In contrast, thrombin failed to increase ROS in Nox1 null SMCs. Previous studies have identified Src and mitogen-activated protein kinases as key redox-dependent regulatory proteins in thrombin-stimulated responses. Five minutes following thrombin stimulation, both Src and ERK1/2 phosphorylation were significantly decreased in Nox1 null SMCs compared with normal SMCs, measured by densitometry of Western blots. In addition, in response to thrombin, epidermal growth factor receptor (EGFR) phosphorylation was reduced in Nox1 null VSMCs. Conditioned media was collected 24 hours after cells were treated with thrombin and MMP-9 activity measured by gelatin zymography. Thrombin increased MMP-9 more than twofold in control cells; however, thrombin failed to increase MMP-9 activity in Nox1 null cells. Using a wound-scratch assay, the number and distance of cells migrating into the injured area were markedly reduced in SMCs deficient in Nox1. In conclusion, the Nox1 subunit of NADPH oxidase is required by SMCs for thrombin-dependent activation of MMP-9 and cell migration. In addition, Nox1 generation of ROS participates in phosphorylation of Src and of ERK1/2. These findings suggest that Nox1 may play an important role in the pathogenesis of vascular disease.

Bacillus anthracis synthesizes two complex structures, a peptidoglycan cell wall and poly-γ-D-glutamic acid (PDGA) capsule, which require an accessible pool of D-glutamate. The mechanisms, however, underlying the establishment of accessible pools of D-glutamate for B. anthracis are poorly understood. B. anthracis harbors two genes, racE1 and racE2, which are each predicted to encode a glutamate racemase capable of converting L-glutamate to D-glutamate. However, the respective roles, if any, of RacE1 or RacE2 in catalyzing the racemization of D-glutamate to L-glutamate have not been investigated. The objective of this study was to compare the in vitro properties of the racE1 and racE2 gene products, with the explicit purpose of establishing whether either or both of these proteins have the capacity to catalyze the conversion of L-glutamate to D-glutamate. racE1 or racE2 were cloned from B. anthracis Sterne 7702 and expressed as recombinant proteins in Escherichia coli. Each protein was purified to homogeneity. We developed an assay based on circular dichroism for directly monitoring glutamate racemase activity. Both RacE1 and RacE2 exhibited detectable glutamate racemase activity. Characterization of both enzymes revealed similar pH profiles and a lack of dependency for various metal cofactors. However, the catalytic efficiency (kcat /KM) of RacE2 was twice that of RacE1. In addition, RacE2 exists in solution as a dimer, whereas RacE1 exists primarily as a monomer. RacE1 forms a higher-ordered complex in the presence of L-glutamate, whereas the quaternary structure of RacE2 is largely independent of substrate. Collectively, these data indicate that although both racE1 and racE2 encode proteins that catalyze the racemization of L-glutamate to D-glutamate in vitro, differences in the properties of these two enzymes suggest that these two enzymes may have distinct cellular roles.